GSM7472839: SEMseq_cfDNA_rep2; Homo sapiens; OTHER
E. coli C2566 genomic DNA was purified using Monarch Genomic DNA Purification Kit (NEB Ipswich, MA), GM12878 genomic DNA was obtained from Coriell Cell Repositories (Camden, NJ). Input DNA was sheared to 300 bp using the Covaris S2 instrument. Sheared material was transferred to a PCR strip tube to begin library construction. NEBNext DNA Ultra II Reagents (NEB, Ipswich, MA) were used according to the manufacturer's instructions for end repair, A-tailing, and adaptor ligation. The custom made Pyrollo-dC adaptor (NEB Organic Synthesis Division, Ipswich MA), where all dCs are replaced with Pyrollo-dC, was used. The ligated samples were purified using NEBNext Sample Purification Beads  according to the manufacturer's instructions. For ssDNA libraries, prior to deamination the DNA was denatured by heating at 90oC for 10 minutes followed by cooling for 2 minutes on ice. The DNA was then deaminated using 1 µL of PURExpress (NEB, Ipswich, MA) synthesized deaminase protein following manufacturer's recommendations for 1 hour at 37oC. Then 1 µL of Thermolabile Proteinase K (NEB, Ipswich, MA) was added and incubated for 30 min at 37oC followed by 10 min at 60oC. Then the library was amplified using NEBNext Q5U Master Mix (NEB, Ipswich, MA, USA). Paired-end sequencing of 150 cycles (2 x 75 bp) was performed for all the sequencing runs. DNA sequencing of deaminated double-stranded and single-stranded DNA